辣椒游离小孢子培养及其显微观察
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摘要
在辣椒的游离小孢子培养中，试验材料的选择、预处理、培养条件和再生培养等都是培养成功与否的重要影响因素。本试验选用了75-7-3-1、77013、海花3号、83-58和Milord这5个不同的辣椒品种，就基因型、花蕾的选择、预处理方式及培养条件对游离小孢子培养的影响进行研究和探讨，同时对小孢子在离体培养后的发育过程和培养产物的发育过程进行了观察。
本文研究了花蕾外观形态与其内部小孢子发育时期之间的关系，确定了大多数小孢子处于单核靠边期阶段的花蕾形态特征，结果表明当各品种花药内小孢子多数处于单核靠边期时，花瓣通常为白色或是白绿色，花瓣与萼片等长或者稍长，花药颜色为白色或者白中发黄，花药尖端微微发紫。
通过设定不同的预处理方式，研究低温、高温、饥饿、秋水仙素和甘露醇处理这5种预处理方式对小孢子存活率的影响，针对5个品种选择既可以提高小孢子存活率又可以维持小孢子活性的预处理方式。结果表明，各品种对处理的反应不尽相同，而低温和饥饿处理是比较有效的处理，对5个品种有较好的试验结果。
利用正交试验研究培养基类型、碳源、更换糖浓度的时间、活性碳、V_(b1)、谷氨酰胺、水解酪蛋白、AgNO_3、2，4—D、BA、KT、IAA和NAA这13个因素对游离小孢子培养的出胚率和愈伤率的影响，对5个品种的试验结果借助Excel和SAS进行直观分析和方差分析，结果表明：通过对5个品种愈伤率和出胚率结果的直观分析，就参试的13个因素对试验结果的影响程度进行主次的排序，对于愈伤率而言，谷氨酰胺的影响力最大，排在2到5位的依次是碳源、AgNO_3、V_(b1)和水解酪蛋白：对于出胚率的影响程度，前5位的因素是：碳源、AgNO_3、水解酪蛋白、V_(b1)和谷氨酰胺，这5个因素在小孢子发生愈伤和成胚的过程中都起着非常重要的作用。本文还就诸因素各水平对5品种的影响进行了邓肯新复极差分析比较，并借此选择出提高愈伤率和出胚率的最佳配方：同时又对各品种的愈伤率和出胚率进行了T测验，从而得出品种基因型间产生愈合组织和胚状体的差异水平。
在离体培养过程中，对各品种的小孢子的发育途径和对愈伤组织及胚状体的发生、发育过程进行观察。发现在海花3号和83-58中均存在小孢子的B分裂途径，而A分裂途径仅在83-58中可以观察到；另外还观察了胚由球形胚经心形胚、鱼雷胚发育到子叶胚的过程：并就发育过程经常出现的组织发育不全、中途停止发育、愈伤化和畸形苗等问题进行了总结。
对培养中获得的愈伤组织和胚状体进行再生培养，就诱导愈伤分化成苗和胚状体直接培养成苗这2个试验途径进行初步探讨，结果显示胚状体直接成苗的成功率高于需要进行分化诱导的培养途径；胚状体成苗培养的试验中V3培养基比改良MS培养基的成苗率高。成苗后便可对已经发育完全的小植株进行倍性鉴定，并对单倍体植株进行染色体加倍。结果表明：并非所有成苗均为单倍体，但经过胚状体途径成苗的单倍体率要高于经过愈伤组织途径成苗的单倍体率：且在单倍体加倍的成功率上，同样是前者高于后者。
Doubled haploid (DH) plants are valuable in genetics and cytogenesis study, physiological research and breeding work, because the recessive genes are not covered by their dominant alleles. The isolated microspore culture is one way of getting doubled haploid.
Five different pepper cultivars were used in this study, including 75-7-3-1, 77013, Haihua No.3, 83-58 and Milord. The effects of genotype, anther development period, pretreatment and culture condition on microspore culture were studied. At the same time, the development of microspore after isolated and the growing of callus and embryo were observed.
The relationship between characters of bud and stages of microspore was studied. When most microspores at late uninucleate and early bincleate stage, the bud has white or light green petals, the sepal length same to the petals, anther is white or light yellow and tip of anther is light purple. The length of bud at this stage in most cultivars was above 4 mm, but the size of buds was different among different cultivars.
The pretreatment effect on survival rate of microspore was studied by 5 kinds of pretreatment including 4 C, 36 C, no sugar treatment, Mannitol treatment and colchicine treatment. At the same time, the efficient pretreatment that elevating and maintaining survive rate of microspore was chosen for condition test. The result showed that different cultivars have different feedback after different pretreatment, 4 C and no sugar treatment have better effect on survive rate for 5 cultivars then control.
The effects of 13 culture factors on isolated microspore culture are checked in this test through orthogonal tests. 13 factors include type of medium, sugar, time of sugar treatment, active charcoal, sliver nitrate, Vb1, lactabumin hydrolysate, glutamine, 2,4-D, KT, BA, IAA and NAA. Directly review analysis with Excel and variance analysis with SAS show that the sugar, sliver nitrate, lactabumin hydrolysate, Vb1 and glutamine are most important culture factors for the forming of callus and embryo. The mediums that benefit for the microspore development are found by Dun Can's new multiple rang test analysis, but the optimum culture medium varied with genotype. The different of callus and embryo rate between genotype were evaluated by T test.
The development of microspore, callus and embryo are observed during culturing. The B division way is discovered in Haihua No.3 and 83-58, A division way is only discovered in 83-58. The course from ball stage of embryo to plantlet is also observed. The abnormal embryo development are summarized, some embryo developed into abnormal cotyledon, some stopped growing after formed 2 cotyledons because the plantlet lose growing point in the earlier stage, and others stop developing on the stage of embryo.
In this study two different cultivation ways are checked for microspore to plantlet. One is that microspore develop directly into plantlet through embryo, the second is that microspore induce callus forming new growing point. The result shows that the successes rate of callus cultivation that from embryo directly to plantlet are higher than the other, and the V3 medium is better than improved MS medium for embryo cultivation. Finally the chromosome number of plantlet is identified. Colchicine is used to double the chromosome number, but all haploid plant are not doubled. The success rate of haploid and chromosome doubling for plantlet from embryo is higher than the plantle from callus.
本文研究了花蕾外观形态与其内部小孢子发育时期之间的关系，确定了大多数小孢子处于单核靠边期阶段的花蕾形态特征，结果表明当各品种花药内小孢子多数处于单核靠边期时，花瓣通常为白色或是白绿色，花瓣与萼片等长或者稍长，花药颜色为白色或者白中发黄，花药尖端微微发紫。
通过设定不同的预处理方式，研究低温、高温、饥饿、秋水仙素和甘露醇处理这5种预处理方式对小孢子存活率的影响，针对5个品种选择既可以提高小孢子存活率又可以维持小孢子活性的预处理方式。结果表明，各品种对处理的反应不尽相同，而低温和饥饿处理是比较有效的处理，对5个品种有较好的试验结果。
利用正交试验研究培养基类型、碳源、更换糖浓度的时间、活性碳、V_(b1)、谷氨酰胺、水解酪蛋白、AgNO_3、2，4—D、BA、KT、IAA和NAA这13个因素对游离小孢子培养的出胚率和愈伤率的影响，对5个品种的试验结果借助Excel和SAS进行直观分析和方差分析，结果表明：通过对5个品种愈伤率和出胚率结果的直观分析，就参试的13个因素对试验结果的影响程度进行主次的排序，对于愈伤率而言，谷氨酰胺的影响力最大，排在2到5位的依次是碳源、AgNO_3、V_(b1)和水解酪蛋白：对于出胚率的影响程度，前5位的因素是：碳源、AgNO_3、水解酪蛋白、V_(b1)和谷氨酰胺，这5个因素在小孢子发生愈伤和成胚的过程中都起着非常重要的作用。本文还就诸因素各水平对5品种的影响进行了邓肯新复极差分析比较，并借此选择出提高愈伤率和出胚率的最佳配方：同时又对各品种的愈伤率和出胚率进行了T测验，从而得出品种基因型间产生愈合组织和胚状体的差异水平。
在离体培养过程中，对各品种的小孢子的发育途径和对愈伤组织及胚状体的发生、发育过程进行观察。发现在海花3号和83-58中均存在小孢子的B分裂途径，而A分裂途径仅在83-58中可以观察到；另外还观察了胚由球形胚经心形胚、鱼雷胚发育到子叶胚的过程：并就发育过程经常出现的组织发育不全、中途停止发育、愈伤化和畸形苗等问题进行了总结。
对培养中获得的愈伤组织和胚状体进行再生培养，就诱导愈伤分化成苗和胚状体直接培养成苗这2个试验途径进行初步探讨，结果显示胚状体直接成苗的成功率高于需要进行分化诱导的培养途径；胚状体成苗培养的试验中V3培养基比改良MS培养基的成苗率高。成苗后便可对已经发育完全的小植株进行倍性鉴定，并对单倍体植株进行染色体加倍。结果表明：并非所有成苗均为单倍体，但经过胚状体途径成苗的单倍体率要高于经过愈伤组织途径成苗的单倍体率：且在单倍体加倍的成功率上，同样是前者高于后者。
Doubled haploid (DH) plants are valuable in genetics and cytogenesis study, physiological research and breeding work, because the recessive genes are not covered by their dominant alleles. The isolated microspore culture is one way of getting doubled haploid.
Five different pepper cultivars were used in this study, including 75-7-3-1, 77013, Haihua No.3, 83-58 and Milord. The effects of genotype, anther development period, pretreatment and culture condition on microspore culture were studied. At the same time, the development of microspore after isolated and the growing of callus and embryo were observed.
The relationship between characters of bud and stages of microspore was studied. When most microspores at late uninucleate and early bincleate stage, the bud has white or light green petals, the sepal length same to the petals, anther is white or light yellow and tip of anther is light purple. The length of bud at this stage in most cultivars was above 4 mm, but the size of buds was different among different cultivars.
The pretreatment effect on survival rate of microspore was studied by 5 kinds of pretreatment including 4 C, 36 C, no sugar treatment, Mannitol treatment and colchicine treatment. At the same time, the efficient pretreatment that elevating and maintaining survive rate of microspore was chosen for condition test. The result showed that different cultivars have different feedback after different pretreatment, 4 C and no sugar treatment have better effect on survive rate for 5 cultivars then control.
The effects of 13 culture factors on isolated microspore culture are checked in this test through orthogonal tests. 13 factors include type of medium, sugar, time of sugar treatment, active charcoal, sliver nitrate, Vb1, lactabumin hydrolysate, glutamine, 2,4-D, KT, BA, IAA and NAA. Directly review analysis with Excel and variance analysis with SAS show that the sugar, sliver nitrate, lactabumin hydrolysate, Vb1 and glutamine are most important culture factors for the forming of callus and embryo. The mediums that benefit for the microspore development are found by Dun Can's new multiple rang test analysis, but the optimum culture medium varied with genotype. The different of callus and embryo rate between genotype were evaluated by T test.
The development of microspore, callus and embryo are observed during culturing. The B division way is discovered in Haihua No.3 and 83-58, A division way is only discovered in 83-58. The course from ball stage of embryo to plantlet is also observed. The abnormal embryo development are summarized, some embryo developed into abnormal cotyledon, some stopped growing after formed 2 cotyledons because the plantlet lose growing point in the earlier stage, and others stop developing on the stage of embryo.
In this study two different cultivation ways are checked for microspore to plantlet. One is that microspore develop directly into plantlet through embryo, the second is that microspore induce callus forming new growing point. The result shows that the successes rate of callus cultivation that from embryo directly to plantlet are higher than the other, and the V3 medium is better than improved MS medium for embryo cultivation. Finally the chromosome number of plantlet is identified. Colchicine is used to double the chromosome number, but all haploid plant are not doubled. The success rate of haploid and chromosome doubling for plantlet from embryo is higher than the plantle from callus.
引文
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